Follicle-stimulating hormone and insulin-like growth factor-I attenuate apoptosis in cultured porcine granulosa cells.
Thymocytes and granulosa cells (GC) were cultured up to 48 h to determine the effect of dexamethasone (DEX) on apoptosis in culture (in RPMI containing 10% fetal bovine serum [FBS] at 38 degrees C in a 5% CO2:95% air atmosphere). In experiment 2, GC were cultured for 24 h at a density of 0.5 x 10(6) cells/0.5 ml in Dulbecco's Modified Eagle's medium:Hams F-12 (1:1) containing 1% FBS to determine whether porcine FSH and insulin-like growth factor-I (IGF-I) attenuated apoptosis and to compare two methods of measuring apoptosis: 1) flow cytometry of dispersed cells for subdiploid DNA fluorescence and 2) densitometry of internucleosomal DNA fragments. The percentages of apoptotic (%A0) thymocytes and GC increased (p < 0.01) during 48 h of culture. Compared to no DEX, 0.1 or 1.0 microM DEX in thymocytes caused a 33% further increase (p < 0.01) in %A0 cells but had no effect in GC. In experiment 2, apoptosis, measured by %A0 GC and amount of internucleosomal fragments, decreased (p < 0.01) by 50% during culture in the presence of FSH (4 NIH-S1 mU/ml) or IGF-I (50 ng/ml); results from these techniques were correlated (r = 0.829, n = 44, p < or = 0.0001). We conclude that 1) porcine GC and thymocytes undergo spontaneous apoptosis in culture, 2) two methods of analyzing apoptosis were in excellent agreement, and 3) FSH and IGF-I attenuated spontaneous apoptosis in cultured porcine GC.
Guthrie
H D
HD
Germplasm and Gamete Physiology Laboratory, Agricultural Research Service, USDA, Beltsville, Maryland 20705, USA. dave@ggpl.arsusda.gov
Garrett
W M
WM
Cooper
B S
BS
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D005640:Follicle Stimulating Hormone / Q000494:pharmacology*; D006107:Granulosa Cells / Q000187:drug effects*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D009707:Nucleosomes / Q000502:physiology; D011374:Progesterone / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013552:Swine; D013601:T-Lymphocytes / Q000187:drug effects
D009707:Nucleosomes; D011374:Progesterone; D007334:Insulin-Like Growth Factor I; D005640:Follicle Stimulating Hormone; D004247:DNA